It has been known for some time that general protein export in bacteria and secretion of proteins into the endoplasmic reticulum of eukaryotes depend on the presence of a signal sequence within the primary structure of the protein to be translocated. Signal sequences are ubiquitous and, in general, will function in heterologous systems. Understanding the function of this intragenic export signal is central to elucidating the biochemical pathway of the export process. this proposal describes an approach to studying signal sequence structure and function with the long-term objective of understanding the mechanistic details of protein translocation across biological membranes. Reports over recent years have suggested that the signal sequence is involved in multiple steps in the export process in bacteria. The aim of this proposal is to sort out these steps and to understand how they related to the cellular export machinery. As a model system, the outer membrane LamB protein of E. coli will be studied using a combination of genetic and biochemical approaches. Several LamB signal sequence mutants have been chosen for study: Both LamB6S and 16E have been shown to result in low LamB levels, and LamB16E also has an export defect. These mutants will be used to study the question of whether or not translation arrest occurs during export in E. coli. lamB14D is a point mutation in which export is blocked prior to formation of an export intermediate (et-LamB). Suppressors of this mutations will be studied to see if they restore the ability of lamB14D to reach the et-LamB stage of export. Preliminary data indicate that lamB19R blocks export after the et-LamB stage. A selection for revertants of lamB19R will be carried out to obtain extragenic suppressors. Such a selection may reveal previously-unknown export components. Understanding the mechanism of protein export in E. coli may lead to the development of new antibiotics, and improvement of he technology for expressing recombinant biological molecules in bacteria for pharmaceutical use.